Mutation detection assay

ABSTRACT

A method of sample analysis is provided. In certain embodiments, the method involves: a) amplifying a product from a sample that comprises both wild type copies of a genomic locus and mutant copies of the genomic locus that have a point mutation relative to said wild type copies of the genomic locus, to produce an amplified sample, where: i. the amplifying is done using a first primer and a second primer; and ii. the first primer comprises a 3′ terminal nucleotide that base pairs with the point mutation and also comprises a nucleotide sequence that is fully complementary to a sequence in the locus with the exception of a single base mismatch within 6 bases of the 3′ terminal nucleotide; and b) detecting the presence of said product in said amplified sample using a flap assay that employs an invasive oligonucleotide. A kit for performing the method is also provided.

CROSS-REFERENCING

This application is a continuation of U.S. application Ser. No.16/569,533, filed on Sep. 12, 2019, issued as U.S. Pat. No. 11,091,812,which is a continuation of U.S. application Ser. No. 15/987,793, filedon May 23, 2018, now issued as U.S. Pat. No. 10,450,614, which is acontinuation of U.S. application Ser. No. 15/165,853, filed on May 26,2016, now issued as U.S. Pat. No. 10,000,817, which is a continuation ofU.S. application Ser. No. 14/228,058, filed on Mar. 27, 2014, now issuedas U.S. Pat. No. 9,376,721, which is a divisional of U.S. applicationSer. No. 14/214,508, filed on Mar. 14, 2014, now issued as U.S. Pat. No.9,024,006, which application is a continuation of U.S. application Ser.No. 12/946,752, filed on Nov. 15, 2010, now issued as U.S. Pat. No.8,715,937, which applications are incorporated by reference herein.

BACKGROUND

Several point mutations in the human genome have a direct associationwith a disease. For example, several germline KRAS mutations have beenfound to be associated with Noonan syndrome (Schubbert et al. Nat.Genet. 2006 38: 331-6) and cardio-facio-cutaneous syndrome (Niihori etal. Nat. Genet. 2006 38: 294-6). Likewise, somatic KRAS mutations arefound at high rates in leukemias, colorectal cancer (Burmer et al. Proc.Natl. Acad. Sci. 1989 86: 2403-7), pancreatic cancer (Almoguera et al.Cell 1988 53: 549-54) and lung cancer (Tam et al. Clin. Cancer Res. 200612: 1647-53). Many point mutations in the human genome have no apparentcausative association with a disease.

Methods for the detection of point mutations may be used, for example,to provide a diagnostic for diseases that are associated with the pointmutations.

SUMMARY

A method of sample analysis is provided. In certain embodiments, themethod involves: a) amplifying a product from a sample that comprisesboth wild type copies of a genomic locus and mutant copies of thegenomic locus that have a point mutation relative to said wild typecopies of the genomic locus, to produce an amplified sample, where: i.the amplifying is done using a first primer and a second primer; and ii.the first primer comprises a 3′ terminal nucleotide that base pairs withpoint mutation and also comprises a nucleotide sequence that is fullycomplementary to a sequence in the locus with the exception of a singlebase mismatch within 6 bases of the 3′ terminal nucleotide; and b)detecting the presence of said product in said amplified sample using aflap assay that employs an invasive oligonucleotide. A kit forperforming the method is also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates some of the general principles of aflap assay.

FIG. 2 schematically illustrates one embodiment of the subject method.

FIGS. 3 to 7 each provide data that is described in greater detail inthe Examples section of this application.

DEFINITIONS

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in liquid form,containing one or more analytes of interest.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, are functionalized as ethers, amines, or the likes.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, up to about10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotidesor ribonucleotides, and may be produced enzymatically or synthetically(e.g., PNA as described in U.S. Pat. No. 5,948,902 and the referencescited therein) which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. Naturally-occurring nucleotides include guanine,cytosine, adenine and thymine (G, C, A and T, respectively).

The term “nucleic acid sample,” as used herein denotes a samplecontaining nucleic acid.

The term “target polynucleotide,” as used herein, refers to apolynucleotide of interest under study. In certain embodiments, a targetpolynucleotide contains one or more target sites that are of interestunder study.

The term “oligonucleotide” as used herein denotes a single strandedmultimer of nucleotides of from about 2 to 200 nucleotides.Oligonucleotides may be synthetic or may be made enzymatically, and, insome embodiments, are 10 to 50 nucleotides in length. Oligonucleotidesmay contain ribonucleotide monomers (i.e., may be oligoribonucleotides)or deoxyribonucleotide monomers. An oligonucleotide may be 10 to 20, 11to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100to 150 or 150 to 200 nucleotides in length, for example.

The term “duplex,” or “duplexed,” as used herein, describes twocomplementary polynucleotides that are base-paired, i.e., hybridizedtogether.

The term “primer” as used herein refers to an oligonucleotide that has anucleotide sequence that is complementary to a region of a targetpolynucleotide. A primer binds to the complementary region and isextended, using the target nucleic acid as the template, under primerextension conditions. A primer may be in the range of about 15 to about50 nucleotides although primers outside of this length may be used. Aprimer can be extended from its 3′ end by the action of a polymerase. Anoligonucleotide that cannot be extended from it 3′ end by the action ofa polymerase is not a primer.

The term “extending” as used herein refers to any addition of one ormore nucleotides to the end of a nucleic acid, e.g. by ligation of anoligonucleotide or by using a polymerase.

The term “amplifying” as used herein refers to generating one or morecopies of a target nucleic acid, using the target nucleic acid as atemplate.

The term “denaturing,” as used herein, refers to the separation of anucleic acid duplex into two single strands.

The terms “determining”, “measuring”, “evaluating”, “assessing,”“assaying,” “detecting,” and “analyzing” are used interchangeably hereinto refer to any form of measurement, and include determining if anelement is present or not. These terms include both quantitative and/orqualitative determinations. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

The term “using” has its conventional meaning, and, as such, meansemploying, e.g., putting into service, a method or composition to attainan end.

As used herein, the term “T_(m)” refers to the melting temperature of anoligonucleotide duplex at which half of the duplexes remain hybridizedand half of the duplexes dissociate into single strands. The T_(m) of anoligonucleotide duplex may be experimentally determined or predictedusing the following formula T_(m)=81.5+16.6(log₁₀ [Na⁺])+0.41 (fractionG+C)−(60/N), where N is the chain length and [Na⁺] is less than 1 M. SeeSambrook and Russell (2001; Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y., ch. 10).Other formulas for predicting T_(m) of oligonucleotide duplexes existand one formula may be more or less appropriate for a given condition orset of conditions.

As used herein, the term “T_(m)-matched” refers to a plurality ofnucleic acid duplexes having T_(m)s that are within a defined range,e.g., within 5° C. or 10° C. of each other.

As used herein, the term “reaction mixture” refers to a mixture ofreagents that are capable of reacting together to produce a product inappropriate external conditions over a period of time. A reactionmixture may contain PCR reagents and flap cleavage reagents, forexample, the recipes for which are independently known in the art.

The term “mixture”, as used herein, refers to a combination of elements,that are interspersed and not in any particular order. A mixture isheterogeneous and not spatially separable into its differentconstituents. Examples of mixtures of elements include a number ofdifferent elements that are dissolved in the same aqueous solution, or anumber of different elements attached to a solid support at random or inno particular order in which the different elements are not spatiallydistinct. A mixture is not addressable. To illustrate by example, anarray of spatially separated surface-bound polynucleotides, as iscommonly known in the art, is not a mixture of surface-boundpolynucleotides because the species of surface-bound polynucleotides arespatially distinct and the array is addressable.

As used herein, the term “PCR reagents” refers to all reagents that arerequired for performing a polymerase chain reaction (PCR) on a template.As is known in the art, PCR reagents essentially include a first primer,a second primer, a thermostable polymerase, and nucleotides. Dependingon the polymerase used, ions (e.g., Mg²⁺) may also be present. PCRreagents may optionally contain a template from which a target sequencecan be amplified.

As used herein, the term “flap assay” refers to an assay in which a flapoligonucleotide is cleaved in an overlap-dependent manner by a flapendonuclease to release a flap that is then detected. The principles offlap assays are well known and described in, e.g., Lyamichev et al.(Nat. Biotechnol. 1999 17:292-296), Ryan et al (Mol. Diagn. 19994:135-44) and Allawi et al (J Clin Microbiol. 2006 44: 3443-3447). Forthe sake of clarity, certain reagents that are employed in a flap assayare described below. The principles of a flap assay are illustrated inFIG. 1 . In the flap assay shown in FIG. 1 , an invasive oligonucleotide2 and flap oligonucleotide 4 are hybridized to target 6 to produce afirst complex 8 that contains a nucleotide overlap at position 10. Firstcomplex 8 is a substrate for flap endonuclease. Flap endonuclease 12cleaves flap oligonucleotide 4 to release a flap 14 that hybridizes withFRET cassette 16 that contains a quencher “Q” and a nearby quenchedflourophore “R” that is quenched by the quencher Q. Hybridization offlap 14 to FRET cassette 16 results in a second complex 18 that containsa nucleotide overlap at position 20. The second complex is also asubstrate for flap endonuclease. Cleavage of FRET cassette 16 by flapendonuclease 12 results in release of the fluorophore 22, which producesa fluorescent signal. These components are described in greater detailbelow.

As used herein, the term “invasive oligonucleotide” refers to anoligonucleotide that is complementary to a region in a target nucleicacid. The 3′ terminal nucleotide of the invasive oligonucleotide may ormay not base pair a nucleotide in the target (e.g., which may be thesite of a SNP or a mutation, for example).

As used herein, the term “flap oligonucleotide” refers to anoligonucleotide that contains a flap region and a region that iscomplementary to a region in the target nucleic acid. The targetcomplementary regions on the invasive oligonucleotide and the flapoligonucleotide overlap by a single nucleotide such that, when they areannealed to the target nucleic acid, the complementary sequencesoverlap. As is known, if: a) the 3′ terminal nucleotide of the invasivenucleotide and b) the nucleotide that overlaps with that nucleotide inthe flap oligonucleotide both base pair with a nucleotide in the targetnucleic acid, then a particular structure is formed. This structure is asubstrate for an enzyme, defined below as a flap endonuclease, thatcleaves the flap from the target complementary region of the flapoligonucleotide. If the 3′ terminal nucleotide of the invasiveoligonucleotide does not base pair with a nucleotide in the targetnucleic acid, or if the overlap nucleotide in the flap oligonucleotidedoes not base pair with a nucleotide in the target nucleic acid, thecomplex is not a substrate for the enzyme and there is little or nocleavage.

The term “flap endonuclease” or “FEN” for short, as used herein, refersto a class of nucleolytic enzymes that act as structure specificendonucleases on DNA structures with a duplex containing a singlestranded 5′ overhang, or flap, on one of the strands that is displacedby another strand of nucleic acid, i.e., such that there are overlappingnucleotides at the junction between the single and double-stranded DNA.FENs catalyze hydrolytic cleavage of the phosphodiester bond at thejunction of single and double stranded DNA, releasing the overhang, orthe flap. Flap endonucleases are reviewed by Ceska and Savers (TrendsBiochem. Sci. 1998 23:331-336) and Liu et al (Annu. Rev. Biochem. 200473: 589-615). FENs may be individual enzymes, multi-subunit enzymes, ormay exist as an activity of another enzyme or protein complex, e.g., aDNA polymerase. A flap endonuclease may be thermostable.

As used herein, the term “cleaved flap” refers to a single-strandedoligonucleotide that is a cleavage product of a flap assay.

As used herein, the term “FRET cassette” refers to a hairpinoligonucleotide that contains a fluorophore moiety and a nearby quenchermoiety that quenches the fluorophore. Hybridization of a cleaved flapwith a FRET cassette produces a secondary substrate for the flapendonuclease. Once this substrate is formed, the 5′fluorophore-containing base is cleaved from the cassette, therebygenerating a fluorescence signal.

As used herein, the term “flap assay reagents” refers to all reagentsthat are required for performing a flap assay on a substrate. As isknown in the art, flap assays include an invasive oligonucleotide, aflap oligonucleotide, a flap endonuclease and a FRET cassette, asdescribed above. Flap assay reagents may optionally contain a target towhich the invasive oligonucleotide and flap oligonucleotide bind.

As used herein, the term “genomic locus” refers to a defined region in agenome. A genomic locus exists at the same location in the genomes ofdifferent cells from the same individual, or in different individuals. Agenomic locus in one cell or individual may have a nucleotide sequencethat is identical or very similar (i.e., more than 99% identical) to thesame genomic locus in a different cell or individual. The difference innucleotide sequence between the same locus in different cells orindividuals may be due to one or more nucleotide substitutions. A SNP(single nucleotide polymorphism) is one type of point mutation thatoccurs at the same genomic locus between different individuals in apopulation. Point mutations may be somatic in that they occur betweendifferent cells in the same individual. A genomic locus mutation may bedefined by genomic coordinates, by name, or using a symbol.

As used herein, a “site of a mutation” refers to the position of anucleotide substitution in a genomic locus. Unless otherwise indicated,the site of a mutation in a nucleic acid can have a mutant allele orwild type allele of a mutation. The site of a mutation may be defined bygenomic coordinates, or coordinates relative to the start codon of agene (e.g., in the case of the “KRAS G35T mutation”).

As used herein, the term “point mutation” refers to the identity of thenucleotide present at a site of a mutation in the mutant copy of agenomic locus. The nucleotide may be on either strand of a doublestranded DNA molecule.

As used herein, the term “wild type”, with reference a genomic locus,refers to the alleles of a locus that contain a wild type sequence. Inthe case of locus containing a SNP, the wild type sequence may containthe predominant allele of the SNP.

As used herein, the term “mutant”, with reference to a genomic locus,refers to the alleles of a locus that contain a mutant sequence. In thecase of a locus containing a SNP, the mutant sequence may contain aminor allele of the SNP. The mutant allele of a genomic locus maycontain a nucleotide substitution that is not silent in that it thateither alters the expression of a protein or changes the amino acidsequence of a protein, which causes a phenotypic change (e.g., acancer-related phenotype) in the cells that are heterozygous orhomozygous for the mutant sequence relative to cells containing the wildtype sequence. Alternatively, the mutant allele of a genomic locus maycontain a nucleotide substitution that is silent.

As used herein, the term “corresponds to” and grammatical equivalentsthereof in the context of, for example, a nucleotide in anoligonucleotide that corresponds to a site of a mutation, is intended toidentify the nucleotide that is correspondingly positioned relative to(i.e., positioned across from) a site of a mutation when two nucleicacids (e.g., an oligonucleotide and genomic DNA containing the mutation)are hybridized. Again, unless otherwise indicated (e.g., in the case ofa nucleotide that “does not base pair” or “base pairs” with a pointmutation) a nucleotide that corresponds to a site of a mutation may basepair with either the mutant or wild type allele of a sequence.

A sample that comprises “both wild type copies of a genomic locus andmutant copies of the genomic locus” and grammatical equivalents thereof,refers to a sample that contains multiple DNA molecules of the samegenomic locus, where the sample contains both wild type copies of thegenomic locus (which copies contain the wild type allele of the locus)and mutant copies of the same locus (which copies contain the mutantallele of the locus). In this context, the term “copies” is not intendedto mean that the sequences were copied from one another. Rather, theterm “copies” in intended to indicate that the sequences are of the samelocus in different cells or individuals.

As used herein the term “nucleotide sequence” refers to a contiguoussequence of nucleotides in a nucleic acid. As would be readily apparent,number of nucleotides in a nucleotide sequence may vary greatly. Inparticular embodiments, a nucleotide sequence (e.g., of anoligonucleotide) may be of a length that is sufficient for hybridizationto a complementary nucleotide sequence in another nucleic acid. In theseembodiments, a nucleotide sequence may be in the range of at least 10 to50 nucleotides, e.g., 12 to 20 nucleotides in length, although lengthsoutside of these ranges may be employed in many circumstances.

As used herein the term “fully complementary to” in the context of afirst nucleic acid that is fully complementary to a second nucleic acidrefers to a case when every nucleotide of a contiguous sequence ofnucleotides in a first nucleic acid base pairs with a complementarynucleotide in a second nucleic acid. As will be described below, anucleic acid may be fully complementary to another sequence “with theexception of a single base mismatch”, meaning that the sequences areotherwise fully complementary with the exception of a single basemismatch (i.e., a single nucleotide that does not base pair with thecorresponding nucleotide in the other nucleic acid).

As used herein the term a “primer pair” is used to refer to two primersthat can be employed in a polymerase chain reaction to amplify a genomiclocus. A primer pair may in certain circumstances be referred to ascontaining “a first primer” and “a second primer” or “a forward primer”and “a reverse primer”. Use of any of these terms is arbitrary and isnot intended to indicate whether a primer hybridizes to a top strand orbottom strand of a double stranded nucleic acid.

The nucleotides of an oligonucleotide may be designated by theirposition relative to the 3′ terminal nucleotide of an oligonucleotide.For example, the nucleotide immediately 5′ to the 3′ terminal nucleotideof an oligonucleotide is at the “−1” position, the nucleotideimmediately 5′ to the nucleotide at the −1 position is the “−2”nucleotide, and so on. Nucleotides that are “within 6 bases” of a 3′terminal nucleotide are at the −1, −2, −3, −4, −5 and −6 positionsrelative to the 3′ terminal nucleotide.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In the following description, the skilled artisan will understand thatany of a number of polymerases and flap endonucleases could be used inthe methods, including without limitation, those isolated fromthermostable or hyperthermostable prokaryotic, eukaryotic, or archaealorganisms. The skilled artisan will also understand that the enzymesthat are used in the method, e.g., polymerase and flap endonuclease,include not only naturally occurring enzymes, but also recombinantenzymes that include enzymatically active fragments, cleavage products,mutants, and variants of wild type enzymes.

In further describing the method, the reagent mixture used in the methodwill be described first, followed by a description of the reactionconditions that may be used in the method.

Reaction Mixture

The reaction mixture used in the method generally contains: a)amplification reagents that are sufficient to amplify a target genomiclocus from a nucleic acid sample, where one of the primers used foramplification comprises a 3′ terminal nucleotide that base pairs with apoint mutation in the genomic locus and also comprises a nucleotidesequence that is fully complementary to a sequence in the locus with theexception of a single base mismatch within 6 bases of the 3′ terminalnucleotide; b) flap assay reagents comprising a flap endonuclease and aFRET cassette; c) the nucleic acid sample. The reaction mixture ischaracterized in that it can amplify and detect the presence of mutantcopies of a genomic locus in a background of wild type copies of thelocus in the sample.

Specifically, the reaction mixture used in the method may contain: a)amplification reagents comprising a thermostable polymerase, nucleotides(e.g., dGTP, dATP, dTTP and dCTP), reaction buffer (which includesMg²⁺), a first primer, and a second primer for amplifying a targetgenomic locus from a nucleic acid sample; wherein the first primer: i.comprises a 3′ terminal nucleotide that base pairs with a point mutationin the genomic locus; and ii. comprises a nucleotide sequence that isfully complementary to a sequence in the locus with the exception of asingle base mismatch within 6 bases of the 3′ terminal nucleotide; b)flap assay reagents comprising a flap endonuclease, a FRET cassette anda flap oligonucleotide that comprises a nucleotide that base pairs withsaid point mutation; and c) the nucleic acid sample, wherein the nucleicacid sample comprises both wild type copies of the genomic locus andmutant copies of the genomic locus that have a point mutation relativeto the wild type copies of the genomic locus. The reaction mixture ischaracterized in that it can amplify and detect the presence of saidmutant copies of the genomic locus in the sample.

In certain embodiments and as will be described in greater detail below,the first primer of the amplification reagents may be employed as aninvasive primer in the flap assay reagents. In other embodiments,however, the reaction mixture may optionally contain, an invasiveoligonucleotide that is distinct from the first primer that has a 3′terminal nucleotide that base pairs with the point mutation. Therefore,depending on how the assay is performed (i.e., depending on whether oneof the PCR primers is used as an invasive oligonucleotide in the flapassay) the reaction mix may additionally contain an invasiveoligonucleotide that is distinct from the PCR primers.

The exact identities and concentrations of the reagents present in thereaction mixture may be similar to or the same as those independentlyemployed in PCR and flap cleavage assays, with the exception that thereaction mixture contains Mg²⁺ at a concentration that is higher thanemployed in conventional PCR reaction mixtures (which contain Mg²⁺ at aconcentration of between about 1.8 mM and 3 mM). In certain embodiments,the reaction mixture described herein contains Mg²⁺ at a concentrationof in the range of 4 mM to 10 mM, e.g., 6 mM to 9 mM. Exemplary reactionbuffers and DNA polymerases that may be employed in the subject reactionmixture include those described in various publications (e.g., Ausubel,et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995and Sambrook et al., Molecular Cloning: A Laboratory Manual, ThirdEdition, 2001 Cold Spring Harbor, N.Y.). Reaction buffers and DNApolymerases suitable for PCR may be purchased from a variety ofsuppliers, e.g., Invitrogen (Carlsbad, CA), Qiagen (Valencia, CA) andStratagene (La Jolla, CA). Exemplary polymerases include Taq, Pfu, Pwo,UlTma and Vent, although many other polymerases may be employed incertain embodiments. Guidance for the reaction components suitable foruse with a polymerase as well as suitable conditions for their use isfound in the literature supplied with the polymerase. Primer design isdescribed in a variety of publications, e.g., Diffenbach and Dveksler(PCR Primer, A Laboratory Manual, Cold Spring Harbor Press 1995); R.Rapley, (The Nucleic Acid Protocols Handbook (2000), Humana Press,Totowa, N.J.); Schena and Kwok et al., Nucl. Acid Res. 199018:999-1005). Primer and probe design software programs are alsocommercially available, including without limitation, Primer Detective(ClonTech, Palo Alto, Calif.), Lasergene, (DNASTAR, Inc., Madison,Wis.); and Oligo software (National Biosciences, Inc., Plymouth, Minn.)and iOligo (Caesar Software, Portsmouth, N.H).

Exemplary flap cleavage assay reagents are found in Lyamichev et al.(Nat. Biotechnol. 1999 17:292-296), Ryan et al (Mol. Diagn. 19994:135-44) and Allawi et al (J Clin Microbiol. 2006 44: 3443-3447).Appropriate conditions for flap endonuclease reactions are either knownor can be readily determined using methods known in the art (see, e.g.,Kaiser et al., J. Biol. Chem. 274:21387-94, 1999). Exemplary flapendonucleases that may be used in the method include, withoutlimitation, Thermus aquaticus DNA polymerase I, Thermus thermophilus DNApolymerase I, mammalian FEN-1, Archaeoglobus fulgidus FEN-1,Methanococcus jannaschii FEN-1, Pyrococcus furiosus FEN-1,Methanobacterium thermoautotrophicum FEN-1, Thermus thermophilus FEN-1,CLEAVASE™ (Third Wave, Inc., Madison, Wis.), S. cerevisiae RTH1, S.cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5′-3′exonuclease, Pyroccus horikoshii FEN-1, human exonuclease 1, calf thymus5′-3′ exonuclease, including homologs thereof in eubacteria, eukaryotes,and archaea, such as members of the class II family ofstructure-specific enzymes, as well as enzymatically active mutants orvariants thereof. Descriptions of cleaving enzymes can be found in,among other places, Lyamichev et al., Science 260:778-83, 1993; Eis etal., Nat. Biotechnol. 19:673-76, 2001; Shen et al., Trends in Bio. Sci.23:171-73, 1998; Kaiser et al. J. Biol. Chem. 274:21387-94, 1999; Ma etal., J. Biol. Chem. 275:24693-700, 2000; Allawi et al., J. Mol. Biol.328:537-54, 2003; Sharma et al., J. Biol. Chem. 278:23487-96, 2003; andFeng et al., Nat. Struct. Mol. Biol. 11:450-56, 2004.

In particular embodiments, the reaction mix may contain reagents forassaying multiple (e.g., at least 2, 3, 4 or more) different targetssequences in parallel. In these cases, the reaction mix may containmultiple pairs of PCR primers, multiple different flap oligonucleotideshaving different flaps, and multiple different FRET cassettes fordetecting the different flaps, once they are cleaved. In one embodiment,oligonucleotides in a mixture may have common flaps but differentbinding sequences to allow for, for example, a set of mutations tocleave a common FRET cassette and report a signal where a singlefluorophore is indicative of the presence of a mutation. In thisembodiment, which mutation is present in the sample may be determinedafter the presence of a mutation has identified. Optionally, thereaction may contain multiple invasive oligonucleotides if one of thePCR primers is not used as an invasive oligonucleotide. Upon cleavage ofthe FRET cassettes, multiple distinguishable fluorescent signals may beobserved. The fluorophore may be selected from, e.g.,6-carboxyfluorescein (FAM), which has excitation and emissionwavelengths of 485 nm and 520 nm respectively, Redmond Red, which hasexcitation and emission wavelengths of 578 nm and 650 nm respectivelyand Yakima Yellow, which has excitation and emission wavelengths of 532nm and 569 nm respectively, and Quasor670 which has excitation andemission wavelengths of 644 nm and 670 nm respectively, although manyothers could be employed. In certain cases, at least one of the PCRprimer pairs, flap oligonucleotides and FRET cassettes may be for thedetection of an internal control.

As noted above, one of the PCR primers (arbitrarily designated as the“first” primer), comprises a 3′ terminal nucleotide that base pairs witha point mutation (i.e., the mutant allele) in the genomic locus and alsocomprises a nucleotide sequence that is fully complementary to asequence in the locus with the exception of a single base mismatchwithin 6 bases of the 3′ terminal nucleotide (e.g., at the −1 position,the −2 position, the −3 position, the −4 position, the −5 position orthe −6 position, relative to the 3′ terminal nucleotide). In otherwords, in addition to having a 3′ terminal nucleotide that base pairswith only the mutant allele of the mutation in the genomic locus, theprimer also has a destabilizing mismatch near the 3′ end that neitherbases pairs with the mutant allele or the wild type allele of thegenomic region. Without being limited to any particular theory, thedestabilizing mismatch is believed to destabilize hybridization of the3′ end of the first primer to the wild-type sequence to a greater extendthan mutant sequence, thereby resulting in preferential amplification ofthe mutant sequence. As will be described in greater detail below, thepresence of the product amplified using the first and second primers maybe detected using a flap assay that employs the first primer or anotheroligonucleotide that has the destabilizing mutation and a terminalnucleotide that base pairs with only the mutant allele at the genomiclocus. The use of such a sequence (i.e., a sequence that contains thedestabilizing mutation and a terminal nucleotide that base pairs withonly the mutant allele at the genomic locus) in the detection stepprovides further discrimination between mutant and wild type sequencesin the amplification products. Without being bound to any particulartheory, it is believed that the discrimination between mutant and wildtype largely occurs in the first few rounds of amplification since theamplified sequence (i.e., the amplicon) provides a perfectlycomplementary sequence for the PCR primers to hybridize to. The wildtype sequence should not be amplified, whereas the mutant sequenceshould be efficiently amplified.

The destabilizing mismatch can be done by substituting a nucleotide thatbase pairs with the point mutation with another nucleotide. Thenucleotide that is substituted into the sequence may be another naturalnucleotide (e.g., dG, dA, dT or dC), or, in certain circumstances, amodified nucleotide. In certain embodiments, the 3′ end of the firstprimer may contain more than 1, e.g., 2 or 3, mismatches. In particularembodiments, the type of mismatch (e.g., whether the mismatch is a G:Tmismatch or a C:T mismatch, etc.) used affects a primer's ability todiscriminate between wild type and mutant sequences. In general terms,the order of the stabilities (from most stable to least stable) ofvarious mismatches are as follows:G:T>G:G=A:G>T:G>G:A=T:T>T:C>A:C>C:T>A:A>C:A>C:C (as described in Gaffneyand Jones (Biochemistry 1989 26: 5881-5889)), although the basepairsthat surround the mismatch can affect this order in certaincircumstances (see, e.g., Ke et al Nucleic Acids Res. 199321:5137-5143). The mismatch used may be optimized experimentally toprovide the desired discrimination.

As would be apparent, the various oligonucleotides used in the methodare designed so as to not interfere with each other. For example, inparticular embodiments, the flap oligonucleotide may be capped at its 3′end, thereby preventing its extension. Likewise, in certain embodiments,the invasive oligonucleotide may also be capped at its 3′ end if it isnot used as one of the PCR primers. In particular embodiment, if theinvasive oligonucleotide is not used as one of the PCR primers, then theinvasive oligonucleotide may be present at a concentration that is inthe range of 5% to 50%, e.g., 10% to 40% of the concentration of the PCRprimers. Further, in certain cases, the T_(m)s of the flap portion andthe target complementary regions of the flap oligonucleotide mayindependently be at least 10° C. lower (e.g., 10-20° C. lower) than theT_(m)s of the PCR primers, which results in a) less hybridization of theflap oligonucleotide to the target nucleic acid at higher temperatures(65° C. to 75° C.) and b) less hybridization of any cleaved flap to theFRET cassette at higher temperatures (65° C. to 75° C.), therebyallowing the genomic locus to be amplified by PCR at a temperature atwhich the flap does not efficiently hybridize.

In a multiplex reaction, the primers may be designed to have similarthermodynamic properties, e.g., similar T_(m)s, G/C content, hairpinstability, and in certain embodiments may all be of a similar length,e.g., from 18 to 30 nt, e.g., 20 to 25 nt in length. The other reagentsused in the reaction mixture may also be T_(m) matched.

The assay mixture may be present in a vessel, including withoutlimitation, a tube; a multi-well plate, such as a 96-well, a 384-well, a1536-well plate; and a microfluidic device. In certain embodiments,multiple multiplex reactions are performed in the same reaction vessel.Depending on how the reaction is performed, the reaction mixture may beof a volume of 5 μl to 200 μl, e.g., 10 μl to 100 μl, although volumesoutside of this range are envisioned.

In certain embodiments, a subject reaction mix may further contain anucleic acid sample. In particular embodiments, the sample may containgenomic DNA or an amplified version thereof (e.g., genomic DNA amplifiedusing the methods of Lage et al, Genome Res. 2003 13: 294-307 orpublished patent application US20040241658, for example). In exemplaryembodiments, the genomic sample may contain genomic DNA from a mammaliancell, such as, a human, mouse, rat, or monkey cell. The sample may bemade from cultured cells or cells of a clinical sample, e.g., a tissuebiopsy, scrape or lavage or cells of a forensic sample (i.e., cells of asample collected at a crime scene). In particular embodiments, thegenomic sample may be from a formalin fixed paraffin embedded (FFPE)sample.

In particular embodiments, the nucleic acid sample may be obtained froma biological sample such as cells, tissues, bodily fluids, and stool.Bodily fluids of interest include but are not limited to, blood, serum,plasma, saliva, mucous, phlegm, cerebral spinal fluid, pleural fluid,tears, lactal duct fluid, lymph, sputum, cerebrospinal fluid, synovialfluid, urine, amniotic fluid, and semen. In particular embodiments, asample may be obtained from a subject, e.g., a human, and it may beprocessed prior to use in the subject assay. For example, the nucleicacid may be extracted from the sample prior to use, methods for whichare known.

For example, DNA can be extracted from stool from any number ofdifferent methods, including those described in, e.g, Coll et al (J. ofClinical Microbiology 1989 27: 2245-2248), Sidransky et al (Science 1992256: 102-105), Villa (Gastroenterology 1996 110: 1346-1353) and Nollau(BioTechniques 1996 20: 784-788), and U.S. Pat. Nos. 5,463,782,7,005,266, 6,303,304 and 5,741,650. Commercial DNA extraction kits forthe extraction of DNA from stool include the QIAamp stool mini kit(QIAGEN, Hilden, Germany), Instagene Matrix (Bio-Rad, Hercules, Calif.),and RapidPrep Micro Genomic DNA isolation kit (Pharmacia Biotech Inc.Piscataway, N.J.), among others.

Method for Sample Analysis

In performing the subject method, the reaction mixture described abovemay be subjected to one or more sets for thermocycling conditions.Exemplary conditions include, for example those described in Allawi etal (J Clin Microbiol. 2006 44: 3443-3447). In one embodiment, thereaction mixture may be subjected to conventional PCR thermocycling(i.e., multiple rounds of denaturation at a temperature of over 90° C.,e.g., at about 95° C., annealing at a temperature of 65° C. to 75° C.and extension at a temperature of 65° C. to 75° C.) followed by a periodat high temperature to denature the themostable polymerase (e.g., about99° C.), and then a period at a temperature that is about 10° C. belowthe extension temperature during which fluorescence is detected.

In other embodiments, the reaction mixture may be subject to cyclingconditions in which an increase in the amount of amplified product(indicated by the amount of fluorescence) can be measured in real-time,where the term “real-time” is intended to refer to a measurement that istaken as the reaction progresses and products accumulate. Themeasurement may be expressed as an absolute number of copies or arelative amount when normalized to a control nucleic acid in the sample.In one real time embodiment, the reaction may be subjected to thethermocycling conditions described in, e.g., Tadokoro (J. Vir. Methods2009 155: 182-186). In this embodiment, the reaction mixture may besubjected to multiple cycles of four steps that include a denaturationstep at a temperature of over 90° C., e.g., at about 95° C., annealingat a temperature in the range of 61° C. to 69° C., flap cleavage at atemperature of 50° C., and extension at a temperature of 72° C. In thisembodiment, fluorescence can be monitored in each cycle to provide areal time measurement of the amount of product that is accumulating inthe reaction mixture.

In an alternative embodiment, the reaction mixture may be subjected tothe following thermocycling conditions: a first set of 5 to 15 (e.g., 8to 12) cycles of: i. a first temperature of at least 90° C.; ii. asecond temperature in the range of 60° C. to 75° C. (e.g., 65° C. to 75°C.); iii. a third temperature in the range of 65° C. to 75° C.; followedby: a second set of 20-50 cycles of: i. a fourth temperature of at least90° C.; ii. a fifth temperature that is at least 10° C. lower than thesecond temperature (e.g., in the range of 50° C. to 55° C.); and iii. asixth temperature in the range of 65° C. to 75° C. No additionalreagents need to be added to the reaction mixture during thethermocycling, e.g., between the first and second sets of cycles. Inparticular embodiments, the thermostable polymerase is not inactivatedbetween the first and second sets of conditions, thereby allowing thetarget to be amplified during each cycle of the second set of cycles. Inparticular embodiments, the second and third temperatures are the sametemperature such that “two step” thermocycling conditions are performed.Each of the cycles may be independently of a duration in the range of 10seconds to 3 minutes, although durations outside of this range arereadily employed. In each cycle of the second set of cycles (e.g., whilethe reaction is in the fifth temperature), a signal generated bycleavage of the flap probe may be measured to provide a real-timemeasurement of the amount of target nucleic acid in the sample.

Some of the principles of the subject of sample analysis method areschematically illustrated in FIG. 2 . With reference to FIG. 2 , themethod includes amplifying product 30 from sample 32 that comprises bothwild type copies of a genomic locus 34 and mutant copies of the genomiclocus 36 that have a point mutation 38 relative to the wild type copiesof the genomic locus 34, to produce an amplified sample. The amplifyingis done using a first primer 40 and a second primer 42, where the firstprimer comprises a 3′ terminal nucleotide 44 that base pairs with thepoint mutation and also comprises a nucleotide sequence that is fullycomplementary to a sequence in the locus with the exception of a singlebase mismatch 46 (i.e., a base that is not complementary to thecorresponding base in the target genomic locus) within 6 bases of 3′terminal nucleotide 44. The presence of product 30 in the amplifiedsample is detected using a flap assay that employs an invasiveoligonucleotide 48 having a 3′ terminal nucleotide that base pairs withthe point mutation. As shown in FIG. 2 , the first primer 40 is employedas the invasive oligonucleotide 48 in the flap assay, although, inalternative embodiments, a second oligonucleotide that is distinct fromthe first primer may be used. As described above and in FIG. 1 , theflap assay relies on the cleavage of complex 32 that contains a flapoligonucleotide 50, invasive oligonucleotide 48 and product 30 by a flapendonuclease (not shown) to release flap 52. Released flap 52 thenhybridizes to FRET cassette 54 to form a second complex that is cleavedby the flap endonuclease to cleave the fluorophore from the complex andgenerate fluorescent signal 56 that can be measured to indicate theamount of product in the amplified sample.

The amount of product in the sample may be normalized relative to theamount of a control nucleic acid present in the sample, therebydetermining a relative amount of the mutant copies in the sample. Insome embodiments, the control nucleic acid may be a different locus tothe genomic locus and, in certain cases, may be detected using a flapassay that employs an invasive oligonucleotide having a 3′ terminalnucleotide that base pairs with the wild type copies of the genomiclocus at the site of the point mutation, thereby detecting the presenceof wild type copies of the genomic locus in said sample. The control maybe measured in parallel with measuring the product in the same reactionmixture or a different reaction mix. If the control is measured in thesame reaction mixture, the flap assay may include further reagents,particularly a second invasive oligonucleotide, a second flap probehaving a second flap and a second FRET cassette that produces a signalthat is distinguishable from the FRET cassette used to detect the mutantsequence. In particular embodiments, the reaction mixture may furthercomprise PCR reagents and flap reagents for amplifying and detecting asecond genomic locus or for detecting a second point mutation in thesame genomic locus.

In certain cases, fluorescence indicating the amount of cleaved flap canbe detected by an automated fluorometer designed to perform real-timePCR having the following features: a light source for exciting thefluorophore of the FRET cassette, a system for heating and coolingreaction mixtures and a fluorometer for measuring fluorescence by theFRET cassette. This combination of features, allows real-timemeasurement of the cleaved flap, thereby allowing the amount of targetnucleic acid in the sample to be quantified. Automated fluorometers forperforming real-time PCR reactions are known in the art and can beadapted for use in this specific assay, for example, the ICYCLER™ fromBio-Rad Laboratories (Hercules, Calif.), the Mx3000P™, the MX3005P™ andthe MX4000™ from Stratagene (La Jolla, Calif.), the ABI PRISM™ 7300,7500, 7700, and 7900 Taq Man (Applied Biosystems, Foster City, Calif.),the SMARTCYCLER™, ROTORGENE 2000™ (Corbett Research, Sydney, Australia)and the GENE XPERT™ System (Cepheid, Sunnyvale, Calif.) and theLIGHTCYCLER™ (Roche Diagnostics Corp., Indianapolis, Ind.). The speed oframping between the different reaction temperatures is not critical and,in certain embodiments, the default ramping speeds that are preset onthermocyclers may be employed.

In certain cases, the method may further involve graphing the amount ofcleavage that occurs in several cycles, thereby providing a real timeestimate of the abundance of the nucleic acid target. The estimate maybe calculated by determining the threshold cycle (i.e., the cycle atwhich this fluorescence increases above a predetermined threshold; the“Ct” value or “Cp” value). This estimate can be compared to a control(which control may be assayed in the same reaction mix as the genomiclocus of interest) to provide a normalized estimate. The thermocyclermay also contain a software application for determining the thresholdcycle for each of the samples. An exemplary method for determining thethreshold cycle is set forth in, e.g., Luu-The et al (Biotechniques 200538: 287-293).

A device for performing sample analysis is also provided. In certainembodiments, the device comprises: a) a thermocycler programmed toperform the above-described method and b) a vessel comprising theabove-described reaction mixture.

Utility

The method described finds use in a variety of applications, where suchapplications generally include sample analysis applications in which thepresence of a target nucleic acid sequence in a given sample isdetected.

In particular, the above-described methods may be employed to diagnose,to predict a response to treatment, or to investigate a cancerouscondition or another mammalian disease, including but not limited to,leukemia, breast carcinoma, prostate cancer, Alzheimer's disease,Parkinsons's disease, epilepsy, amylotrophic lateral schlerosis,multiple sclerosis, stroke, autism, mental retardation, anddevelopmental disorders. Many nucleotide polymorphisms are associatedwith and are thought to be a factor in producing these disorders.Knowing the type and the location of the nucleotide polymorphism maygreatly aid the diagnosis, prognosis, and understanding of variousmammalian diseases. In addition, the assay conditions described hereincan be employed in other nucleic acid detection applications including,for example, for the detection of infectious diseases, viral loadmonitoring, viral genotyping, environmental testing, food testing,forensics, epidemiology, and other areas where specific nucleic acidsequence detection is of use.

In some embodiments, a biological sample may be obtained from a patient,and the sample may be analyzed using the method. In particularembodiments, the method may be employed to identify and/or estimate theamount of mutant copies of a genomic locus that are in a biologicalsample that contains both wild type copies of a genomic locus and mutantcopies of the genomic locus that have a point mutation relative to thewild type copies of the genomic locus. In this example, the sample maycontain at least 100 times (e.g., at least 1,000 times, at least 5,000times, at least 10,000 times, at least 50,000 times or at least 100,000times) more wild type copies of the genomic locus than mutant copiessaid genomic locus.

In these embodiments, the method may be employed to detect an oncogenicmutation (which may be a somatic mutation) in, e.g., PIK3CA, NRAS, KRAS,JAK2, HRAS, FGFR3, FGFR1, EGFR, CDK4, BRAF, RET, PGDFRA, KIT or ERBB2,which mutation may be associated with breast cancer, melanoma, renalcancer, endometrial cancer, ovarian cancer, pancreatic cancer, leukemia,colorectal cancer, prostate cancer, mesothelioma, glioma,medullobastoma, polycythemia, lymphoma, sarcoma or multiple myeloma(see, e.g., Chial 2008 Proto-oncogenes to oncogenes to cancer. NatureEducation 1:1).

In these embodiments, the reaction mixture may contain a first primerand a second primer wherein the first primer comprises a 3′ terminalnucleotide that base pairs with the point mutation. The first primer maybe employed as the invasive oligonucleotide in the second set of cyclesor, in certain cases, there may be a separate invasive oligonucleotidepresent in the reaction mixture that also has a 3′ terminal nucleotidethat base pairs with the point mutation. Since the point mutation in thegenomic locus may have a direct association with cancer, e.g.,colorectal cancer, the subject method may be employed to diagnosepatients with cancer or a pre-cancerous condition (e.g., adenoma etc.),alone, or in combination with other clinical techniques (e.g., aphysical examination, such as, a colonoscopy) or molecular techniques(e.g., immunohistochemical analysis). For example, results obtained fromthe subject assay may be combined with other information, e.g.,information regarding the methylation status of other loci, informationregarding rearrangements or substitutions in the same locus or at adifferent locus, cytogenetic information, information regardingrearrangements, gene expression information or information about thelength of telomeres, to provide an overall diagnosis of cancer or otherdiseases.

In one embodiment, a sample may be collected from a patient at a firstlocation, e.g., in a clinical setting such as in a hospital or at adoctor's office, and the sample may be forwarded to a second location,e.g., a laboratory where it is processed and the above-described methodis performed to generate a report. A “report” as described herein, is anelectronic or tangible document which includes report elements thatprovide test results that may include a Ct value, or Cp value, or thelike that indicates the presence of mutant copies of the genomic locusin the sample. Once generated, the report may be forwarded to anotherlocation (which may the same location as the first location), where itmay be interpreted by a health professional (e.g., a clinician, alaboratory technician, or a physician such as an oncologist, surgeon,pathologist), as part of a clinical diagnosis.

Kits

Also provided are kits for practicing the subject method, as describedabove. The components of the kit may be present in separate containers,or multiple components may be present in a single container. Inparticular embodiments, a kit may comprise: a) PCR reagents that includea first primer and a second primer, wherein the first primer comprises a3′ terminal nucleotide that base pairs with a point mutation in agenomic locus and also comprises a nucleotide sequence that is fullycomplementary to a sequence in the genomic locus with the exception of asingle base mismatch within 6 bases of the 3′ terminal nucleotide; andb) flap assay reagents that include an invasive oligonucleotide having a3′ terminal nucleotide that base pairs with the point mutation. Theparticulars of these reagents are described above. The kit furthercomprises PCR and flap reagents for amplification and detection of acontrol nucleic acid.

In addition to above-mentioned components, the kit may further includeinstructions for using the components of the kit to practice the subjectmethods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate. In addition to theinstructions, the kits may also include one or more control samples,e.g., positive or negative controls analytes for use in testing the kit.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Example 1 KRAS G35A Assay

The assay described below is designed to detect nucleic acid sequencescontaining the KRAS G35A mutation in a background of wild typesequences. For reference, partial nucleotide sequences for the wild typeand G35A mutant alleles of KRAS are shown below.

Partial sequence of amplification region for KRAS, wild type (position35 underlined):

(SEQ ID NO: 1) CATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGA

Partial sequence of amplification region for KRAS, mutant G35A (position35 underlined):

(SEQ ID NO: 2) CATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGATGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGA

In this experiment, the entire burden of specificity for detection anddiscrimination of the G35A mutation rested upon the flap probe. Forwardand reverse primer binding regions were outside of the mutation atposition 35. The invasive oligonucleotide was not the same sequence aseither of the primers. Primers for the amplification of this region were5′-AGGCCTGCTGAAAATGACTG-3′ (SEQ ID NO:3) and5′-TTGTTGGATCATATTCGTCCAC-3′ (SEQ ID NO:4).

The homogeneous detection of the KRAS G35A mutation was accomplished bythe use of an endonuclease cleavable flap probe, an invasiveoligonucleotide probe, a cleavable FRET cassette, and a heat stable flapendonuclease. For the detection of the G35A mutation, the flap probesequence was 5′-CGCCGAGGATGGCGTAGGCA-3′/3C6/(SEQ ID NO:5), where themutant base is shown underlined and the 3′-end is blocked with ahexanediol group in order to inhibit primer extension. The cleaved flapportion, which subsequently binds the FRET cassette, and in turnreleases the fluorophore away from its quencher, includes all of thebases from the 5′-end to the mutation-specific A. The invasiveoligonucleotide used in this example was 5′-TGTGGTAGTTGGAGCTGg-3′ (SEQID NO:6), where the 3′ g is not specific for the G35A. Primers, invasiveoligos, and flap probes were supplied as non-catalog items by IntegratedDNA Technologies (IDT, Coralville, Iowa).

The FRET cassette used was /Red/TCT/Quencher/TCGGCCTTTTGGCCGAGAGACCTCGGCGCG/3C6/(SEQ ID NO:7), where Redmond Red is the fluorescent dye, thequencher is the Eclipse® Dark Quencher, and the 3′-end is blocked with ahexanediol group in order to inhibit primer extension. The FRET cassettewas supplied by Hologic (Madison, Wisconsin). This FRET cassette may bereferred to as Arm 1 Red FRET cassette).

The relative positioning of the primers, invasive probe, and flap probebinding region can be seen as underlined regions on the followingsequence, with the flap probe binding region shown italicized:

(SEQ ID NO: 8) CATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTG ATGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGA

Cycling conditions were 95° C. for 2 min; 50 cycles at 95° C. for 20sec, 50° C. for 1 min, and 70° C. for 30 sec, with a final 40° C. hold.Fluorescent signal acquisition was done at the 50° C. point in thecycle. The PCR reactions were done in LightCycler® 480 Multiwell 96Plates (Roche, Indianapolis) in 10 mM MOPS pH 7.5, with 7.5 mM MgCl₂,and 250 μM dNTPs (Promega, Madison, Wisconsin). Taq polymerase was theiTaq enzyme (BioRad, Hercules, California) and the cleavage enzyme wasCleavase 2.0 (Hologic, Madison, Wisconsin). Forward primer concentrationwas 500 nM, reverse primer concentration was 500 nM, flap probe was at500 nM, invasive oligo probe was at 70 nM, and the FRET cassette wasused at a final concentration of 200 nM. All amplification and detectionwas performed in the LightCycler 480 optical thermocycler (Roche,Indianapolis, Indiana).

Plasmids containing fragments of the KRAS gene, either the wild type orthe mutant, were used to assess the ability of the experimental systemto detect KRAS 35A mutant copies spiked in wild-type copies at 4different levels of the mutant, including 10⁴ mutant copies in 10⁵wild-type copies (1:10), 10³ mutant copies in 10⁵ wild-type copies(1:100), 10² mutant copies in 10⁵ wild-type copies (1:1000), and 10mutant copies in 10⁵ wild-type copies (1:10000).

Data showing kinetic amplification curves and the “crossing point” (Cp;Roche LightCycler 480 Manual, Indianapolis, IN) of the different ratiosof mutant to wild type in the amplification samples are shown in FIG. 3. In these assays, the Cp is calculated as being the point at whichfluorescence rose to 18% of the maximum fluorescence. The design ofprimers, invasive probe, and flap probe used in this example were unableto detect the G35A mutation when there were greater than 100-fold morewild type than mutant forms of the gene.

Example 2 KRAS G35A Assay

As in Example 1, this assay was designed to detect the G35A mutation inthe presence of various levels of the wild type sequence. Mutant andwild type sequence are as described in Example 1 (SEQ ID NO:1 and SEQ IDNO:2). In this assay, the forward primer also served to function as theinvasive oligonucleotide. The 3′ of the forward primer and the pivotalposition in the flap probe both contain the mutant-like A base.

Primers used were 5′-TTGTGGTAGTTGGAGCTGA-3′ (SEQ ID NO:9), where theunderlined 3′ base corresponds to the mutation, and5′-CTATTGTTGGATCATATTCGTC-3′ (SEQ ID NO:10). The flap probe used was thesame as in Example 1, 5′-CGCCGAGGATGGCGTAGGCA-3′/3C6/(SEQ ID NO:5),where the mutant base is shown underlined and the 3′-end is blocked witha hexanediol group in order to inhibit primer extension. Primers andflap probes were supplied as non-catalog items by Integrated DNATechnologies (IDT, Coralville, Iowa).

The relative positioning of the primers, invasive probe, and flap probebinding region can be seen as underlined regions on the followingsequence, with the flap probe binding region shown italicized:

(SEQ ID NO: 11) CATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGA TGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGA

The FRET cassette used was/Red/TCT/Quencher/TCGGCCTTTTGGCCGAGAGACCTCGGCGCG/3C6/(SEQ ID NO: 25)where Redmond Red is the fluorescent dye, the quencher is the Eclipse®Dark Quencher, and the 3′-end is blocked with a hexanediol group inorder to inhibit primer extension. The FRET cassette was supplied byHologic (Madison, Wisconsin).

Cycling conditions were 95° C. for 2 min; 50 cycles at 95° C. for 20sec, 53° C. for 1 min, and 70° C. for 30 sec; and 40° C. to hold.Fluorescent signal acquisition was done at the 53° C. point in thecycle. The PCR reactions were done in LightCycler® 480 Multiwell 96Plates (Roche, Indianapolis) in 10 mM MOPS pH 7.5, with 7.5 mM MgCl₂,and 250 μM dNTPs (Promega, Madison, Wisconsin). Taq polymerase was theiTaq enzyme (BioRad, Hercules, California) and the cleavage enzyme wasCleavase 2.0 (Hologic, Madison, Wisconsin). Forward primer concentrationwas 500 nM, reverse primer concentration was 500 nM, flap probe was at500 nM, and the FRET cassette was used at a final concentration of 200nM. All amplification and detection was performed in the LightCycler 480optical thermocycler (Roche, Indianapolis, Indiana).

Plasmids containing fragments of the KRAS gene, either the wild type orthe mutant, were used to assess the ability of the experimental systemto detect KRAS 35A mutant copies spiked in wild-type copies at 4different levels of the mutant, including 10⁴ mutant copies in 10⁵wild-type copies (1:10), 10³ mutant copies in 10⁵ wild-type copies(1:100), 10² mutant copies in 10⁵ wild-type copies (1:1000), and 10mutant copies in 10⁵ wild-type copies (1:10000).

Data showing kinetic amplification curves and the “crossing point” (Cp;Roche LightCycler 480 Manual, Indianapolis, IN) of the different ratiosof mutant to wild type in the amplification samples are shown in FIG. 4. In these assays, the Cp is calculated as being the point at whichfluorescence rose to 18% of the maximum fluorescence. The design ofprimers and flap probe used in this example were unable to detect theG35A mutation in the presence of wild type, and there is no real doseresponse.

Example 3 KRAS G35A, G35T, and G35C Assays with Secondary Mismatches

In this example, secondary mismatches were added to the forward primer,which also acts as the invasive probe in these cleavage assays. Thesecond mismatch was added to the fourth base, counting from the 3′ endbase that matches the mutation. In all cases, the second mismatch basewas an A, corresponding to a C in the sequence, and thus generating anA:G mispair in the mutant and wild type sequences. The destabilizing Amismatch was employed with primers (which are also the invasive probe)that had 3′ ends with A, T, or C, corresponding to all of the threemutations found at position 35 of KRAS.

The target sequence for the wild type KRAS is as in Example 1 (SEQ IDNO:1). The G35A sequence detected was the one shown in Example 1 (SEQ IDNO:2).

Partial sequence of amplification region for KRAS, mutant G35T (position35 underlined):

(SEQ ID NO: 12) CATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGTTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGA

Partial sequence of amplification region for KRAS, mutant G35C (position35 underlined):

(SEQ ID NO: 13) CATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGCTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGA

The reverse primer used for all three of these mutation detectiondesigns was the same as the one in Example 2,5′-CTATTGTTGGATCATATTCGTC-3′ (SEQ ID NO:10). The forward primer fordetection of the G35A was 5′-TTGTGGTAGTTGGAGATGA-3′ (SEQ ID NO:14) wherethe 3′ end underlined base corresponds to the mutation targeted and theunderlined base four positions from the 3′ end is the destabilizingmismatch intended to augment the effect of the 3′ end mismatch. The flapprobe used for G35A was the same as in Example 1,5′-CGCCGAGGATGGCGTAGGCA-3′/3C6/(SEQ ID NO:5), where the mutant base isshown underlined and the 3′-end is blocked with a hexanediol group inorder to inhibit primer extension.

The forward primer for detection of the G35T was5′-TTGTGGTAGTTGGAGATGT-3′ (SEQ ID NO:15) where the 3′ end underlinedbase corresponds to the mutation targeted and the underlined base fourpositions from the 3′ end is the destabilizing mismatch intended toaugment the effect of the 3′ end mismatch. The flap probe used for G35Twas 5′-CGCCGAGGTTGGCGTAGGCA-3′/3C6/(SEQ ID NO:16), where the mutant baseis shown underlined and the 3′-end is blocked with a hexanediol group inorder to inhibit primer extension.

The forward primer for detection of the G35C was5′-TTGTGGTAGTTGGAGATGC-3′ (SEQ ID NO:17) where the 3′ end underlinedbase corresponds to the mutation targeted and the underlined base, fourpositions from the 3′ end is the destabilizing mismatch intended toaugment the effect of the 3′ end mismatch. The flap probe used for G35Cwas 5′-CGCCGAGGCTGGCGTAGGCA-3′/3C6/(SEQ ID NO:18), where the mutant baseis shown underlined and the 3′-end is blocked with a hexanediol group inorder to inhibit primer extension.

The spatial configuration of primers and flap probe is similar to thatshown in Example 2 (SEQ ID NO:11).

Primers and flap probes were supplied as non-catalog items by IntegratedDNA Technologies (IDT, Coralville, Iowa).

The FRET cassette used, for all of these mutation detection systems used/Red/TCT/Quencher/TCGGCCTTTTGGCCGAGAGACCTCGGCGCG/3C6/(SEQ ID NO:25),where Redmond Red is the fluorophore, the quencher is the Eclipse® DarkQuencher, and the 3′-end is blocked with a hexanediol group in order toinhibit primer extension. The FRET cassette was supplied by Hologic(Madison, Wisconsin).

Cycling conditions were 95° C. for 2 min; 50 cycles at 95° C. for 20sec, 53° C. for 1 min, and 70° C. for 30 sec; and 40° C. to hold.Fluorescent signal acquisition was done at the 53° C. point in thecycle. The PCR reactions were done in LightCycler® 480 Multiwell 96Plates (Roche, Indianapolis) in 10 mM MOPS pH 7.5, with 7.5 mM MgCl₂,and 250 μM dNTPs (Promega, Madison, Wisconsin). Taq polymerase was theiTaq enzyme (BioRad, Hercules, California) and the cleavage enzyme wasCleavase 2.0 (Hologic, Madison, Wisconsin). Forward primer concentrationwas 500 nM, reverse primer concentration was 500 nM, flap probe was at500 nM, and the FRET cassette was used at a final concentration of 200nM. All amplification and detection was performed in the LightCycler 480optical thermocycler (Roche, Indianapolis, Indiana).

Plasmids containing fragments of the KRAS gene, either the wild type orrespectively one of the three mutants, were used to assess the abilityof the experimental system to detect KRAS G35A, T, or C mutant copiesspiked in wild-type copies at 4 different levels of the mutant,including 10⁴ mutant copies in 10⁵ wild-type copies (1:10), 10³ mutantcopies in 10⁵ wild-type copies (1:100), 10² mutant copies in 10⁵wild-type copies (1:1000), and 10 mutant copies in 10⁵ wild-type copies(1:10000).

Data showing kinetic amplification curves and the “crossing point” (Cp;Roche LightCycler 480 Manual, Indianapolis, IN) of the different ratiosof mutant to wild type in the amplification samples are shown in FIG. 5. In these assays, the Cp is calculated as being the point at whichfluorescence rose to 18% of the maximum fluorescence. The design ofprimers and flap probe used in this example could discriminate mutantsequence from wild-type sequence, and its amplification efficiency wasnot suppressed by excessive amounts of wild-type sequences down to about1:100 mutant/wild-type ratio. However, the assay could not reliablydetect mutant sequences at a 1:1000 mutant/wild-type ratio (FIG. 5 ).

Example 4 KRAS G35A, G35T, and G35C Assays with Other SecondaryMismatches

In this example, secondary mismatches were tested, similar to Example 3,except mismatches known to be more destabilizing to base pairing,specifically A-C and C-C mismatches were tested at the penultimateposition, adjacent to the 3′-end mismatch, for G35A and G35T,respectively. The same primer set as used in Example 3 was used fordetection of G35C. In addition, a different flap arm of the flap probewas used as well as a correspondingly paired FRET cassette.

Target sequences for the three mutations are the same as those describedin Example 1 and Example 3. The reverse primer used for all three ofthese mutation detection experiments was the same as the one in Example2, 5′-CTATTGTTGGATCATATTCGTC-3′ (SEQ ID NO:10).

The forward primer for detection of G35C are the same as described inExample 3, 5′-TTGTGGTAGTTGGAGATGC-3′ (SEQ ID NO:17) where the 3′ endunderlined base corresponds to the mutation targeted and the underlinedbase, four positions from the 3′ end is the destabilizing mismatchintended to augment the effect of the 3′ end mismatch.

The forward primer for detection of the G35A was5′-TTGTGGTAGTTGGAGCTAA-3′ (SEQ ID NO:19) where the 3′ end underlinedbase corresponds to the mutation targeted and the penultimate underlinedbase is the destabilizing mismatch intended to augment the effect of the3′ end mismatch. The forward primer for detection of the G35T was5′-TTGTGGTAGTTGGAGCTCT-3′ (SEQ ID NO:20) where the 3′ end underlinedbase corresponds to the mutation targeted and the penultimate underlinedbase is the destabilizing mismatch intended to augment the effect of the3′ end mismatch.

The flap probe for detection of G35A was5′-GACGCGGAGATGGCGTAGGCA-3′/3C6/(SEQ ID NO:21) where the mutant base isshown underlined and the 3′-end is blocked with a hexanediol group inorder to inhibit primer extension. The flap probe for detection of G35Twas 5′-GACGCGGAGTTGGCGTAGGCA-3′/3C6/(SEQ ID NO:22) where the mutant baseis shown underlined and the 3′-end is blocked with a hexanediol group inorder to inhibit primer extension. The flap probe for detection of G35Cwas 5′-GACGCGGAGCTGGCGTAGGCA-3′/3C6/(SEQ ID NO:23) where the mutant baseis shown underlined and the 3′-end is blocked with a hexanediol group inorder to inhibit primer extension.

The FRET cassette used, for the mutation detection in this example was5′-FAM/TCT/Quencher/AGCCGGTTTTCCGGCTGAGACTCCGCGTCCGT-3′/3C6 (SEQ IDNO:24), where FAM is fluorescein, the quencher is the Eclipse® DarkQuencher, and the 3′-end is blocked with a hexanediol group in order toinhibit primer extension. The FRET cassette was supplied by Hologic(Madison, Wisconsin). Cycling conditions were 95° C. for 2 min; 50cycles at 95° C. for 20 sec, 53° C. for 1 min, and 70° C. for 30 sec;and 40° C. to hold. Fluorescent signal acquisition was done at the 53°C. point in the cycle. The PCR reactions were done in LightCycler® 480Multiwell 96 Plates (Roche, Indianapolis) in 10 mM MOPS pH 7.5, with 7.5mM MgCl₂, and 250 μM dNTPs (Promega, Madison, Wisconsin). Taq polymerasewas the iTaq enzyme (BioRad, Hercules, California) and the cleavageenzyme was Cleavase 2.0 (Hologic, Madison, Wisconsin). Forward primerconcentration was 500 nM, reverse primer concentration was 500 nM, flapprobe was at 500 nM, and the FRET cassette was used at a finalconcentration of 200 nM. All amplification and detection was performedin the LightCycler 480 optical thermocycler (Roche, Indianapolis,Indiana).

Plasmids containing fragments of the KRAS gene, either the wild type orrespectively one of the three mutants, were used to assess the abilityof the experimental system to detect KRAS G35A, T, or C mutant copiesspiked in wild-type copies at 4 different levels of the mutant,including 10⁴ mutant copies in 10⁵ wild-type copies (1:10), 10³ mutantcopies in 10⁵ wild-type copies (1:100), 10² mutant copies in 10⁵wild-type copies (1:1000), and 10 mutant copies in 10⁵ wild-type copies(1:10000).

Data showing kinetic amplification curves and the “crossing point” (Cp;Roche LightCycler 480 Manual, Indianapolis, IN) of the different ratiosof mutant to wild type in the amplification samples are shown in FIG. 6. In these assays, the Cp is calculated as being the point at whichfluorescence rose to 18% of the maximum fluorescence. The design ofprimers and flap probe used in this example could discriminate mutantsequence from wild-type sequence, and its amplification efficiency wasnot suppressed by excessive amounts of wild-type sequences. KRAS G35Aand G35C assays could linearly detect mutant sequence down to a ratio of1:1000 mutant/wild-type ratio, and the G35T assay could linearly detectdown to a ratio of 1:100 mutant/wild-type ratio (FIG. 6 ).

Example 5 Additional Experimental Determination of Best SecondaryMismatches for KRAS G35A Detection

In this example, similar to the previous examples, the destabilizingeffect of secondary mismatches in the forward primer, which alsofunctions as the invasive probe was tested using the mutation atposition 35 of KRAS. As in the other examples, the detection of the G35Amutation was assessed at various ratios compared to an excess of thewild type sequence. Target amplification region sequences were the sameas in Example 1 (SEQ ID NO:1 and SEQ ID NO:2). The reverse primer usedfor all three of these mutation detection experiments was the same asthe one in Examples 2, 3, and 4: 5′-CTATTGTTGGATCATATTCGTC-3′ (SEQ IDNO:10).

The flap probe (SEQ ID NO:21) and FRET cassette (SEQ ID NO:24) were thesame as those used for detection of G35A in Example 4. The series offorward probes, which also serve as invasive oligos for the cleavageassay are listed, where the 3′ end underlined base corresponds to themutation targeted and the other underlined base is the destabilizingmismatch intended to augment the effect of the 3′ end mismatch:

KRAS 35A P2C: (SEQ ID NO: 25) 5′-TTGTGGTAGTTGGAGCTCA-3′ KRAS 35A P4A:(SEQ ID NO: 26) 5′-AACTTGTGGTAGTTGGAGATGA-3′ KRAS 35A P6T:(SEQ ID NO: 27) 5′-CTTGTGGTAGTTGGTGCTGA-3′ KRAS 35A P5C: (SEQ ID NO: 28)5′-AACTTGTGGTAGTTGGACCTGA-3′ KRAS 35A P3A: (SEQ ID NO: 29)5′-CTTGTGGTAGTTGGAGCAGA-3′

Cycling conditions were 95° C. for 2 min; 50 cycles at 95° C. for 20sec, 53° C. for 1 min, and 70° C. for 30 sec; and 40° C. to hold.Fluorescent signal acquisition was done at the 50° C. point in thecycle. The PCR reactions were done in LightCycler® 480 Multiwell 96Plates (Roche, Indianapolis) in 10 mM MOPS pH 7.5, with 7.5 mM MgCl₂,and 250 μM dNTPs (Promega, Madison, Wisconsin). Taq polymerase was theiTaq enzyme (BioRad, Hercules, California) and the cleavage enzyme wasCleavase 2.0 (Hologic, Madison, Wisconsin). Forward primer concentrationwas 500 nM, reverse primer concentration was 500 nM, flap probe was at500 nM, and the FRET cassette was used at a final concentration of 200nM. All amplification and detection was performed in the LightCycler 480optical thermocycler (Roche, Indianapolis, Indiana).

Plasmids containing fragments of the KRAS gene, either the wild type orrespectively one of the three mutants, were used to assess the abilityof the experimental system to detect KRAS G35A, T, or C mutant copiesspiked in wild-type copies at 4 different levels of the mutant,including 10⁴ mutant copies in 10⁵ wild-type copies (1:10), 10³ mutantcopies in 10⁵ wild-type copies (1:100), 10² mutant copies in 10⁵wild-type copies (1:1000), and 10 mutant copies in 10⁵ wild-type copies(1:10000).

Data showing kinetic amplification curves and the “crossing point” (Cp;Roche LightCycler 480 Manual, Indianapolis, IN)” of the different ratiosof mutant to wild type in the amplification samples are shown in FIG. 7. In these assays, the Cp is calculated as being the point at whichfluorescence rose to 18% of the maximum fluorescence. Out of the 5primer/invasive probe designs tested, the penultimate mismatch, yieldinga C:C mispair, designated KRAS 35A P2C showed the best ability toquantify mutant in the presence of excess wild type gene sequence,showing a dose response to a level of 1:1000 mutant/wild type (FIG. 7 ).

What is claimed is:
 1. A method of sample analysis comprising: a)amplifying a plurality of products from a sample using a reactionmixture comprising: a first primer pair for amplifying a first genomiclocus; and a second primer pair for amplifying a second genomic locusdifferent from the first genomic locus, wherein the sample comprisesboth wild type copies of the first and second genomic loci and mutantcopies of the first and second genomic loci, wherein the mutant copiesof the first genomic locus have a point mutation relative to the wildtype copies of the first genomic locus, and the mutant copies of thesecond genomic locus have a point mutation relative to the wild typecopies of the second genomic locus, to produce an amplified sample;wherein: i. each of the first and second primer pairs comprises a firstprimer and a second primer; ii. the first primer of the first primerpair comprises a 3′ terminal nucleotide that base pairs with the pointmutation at the first genomic locus and also comprises a nucleotidesequence that is fully complementary to a sequence in the locus with theexception of a single base mismatch within 6 bases of said 3′ terminalnucleotide; and iii. the first primer of the second primer paircomprises a 3′ terminal nucleotide that base pairs with the pointmutation at the second genomic locus and also comprises a nucleotidesequence that is fully complementary to a sequence in the locus with theexception of a single base mismatch within 6 bases of said 3′ terminalnucleotide; and b) detecting the presence of the plurality of productsin the amplified sample using a flap assay that employs: i. the firstprimer of the first primer pair or a first invasive oligonucleotidehaving a 3′ terminal nucleotide that base pairs with the point mutationat the first genomic locus; ii. the first primer of the second primerpair or a second invasive oligonucleotide having a 3′ terminalnucleotide that base pairs with the point mutation at the second genomiclocus; iii. a first flap oligonucleotide that comprises a nucleotidethat base pairs with the point mutation at the first loci; and iv. asecond flap oligonucleotide that comprises a nucleotide that base pairswith the point mutation at the second loci.
 2. The method of claim 1,wherein the flap assay employs the first primer of the first primerpair.
 3. The method of claim 1, wherein the flap assay employs the firstinvasive oligonucleotide.
 4. The method of claim 1, wherein the flapassay employs the first primer of the second primer pair.
 5. The methodof claim 1, wherein the flap assay employs the second invasiveoligonucleotide.
 6. The method of claim 1, wherein the flap assayemploys a first flap probe comprising a first flap and a first FRETcassette and a second flap probe comprising a second flap and a secondFRET cassette that produces a signal that is distinguishable from thefirst FRET cassette.
 7. The method of claim 1, wherein the mismatch inthe first primer of the first primer pair is at position −1, position−2, position −3, position −4 or position −5 relative to said terminalnucleotide.
 8. The method of claim 1, wherein the mismatch in the firstprimer of the second primer pair is at position −1, position −2,position −3, position −4 or position −5 relative to said terminalnucleotide.
 9. The method of claim 1, wherein the sample contains atleast 100 times more wild type copies of the first genomic locus thanmutant copies of the first genomic locus.
 10. The method of claim 1,wherein the sample contains at least 100 times more wild type copies ofthe second genomic locus than mutant copies of the second genomic locus.11. The method of claim 1, further comprising normalizing the amount ofthe plurality of products in the amplified sample relative to the amountof a control nucleic acid present in the sample, thereby determining theamount of the mutant copies of the first and second genomic loci in thesample.
 12. The method of claim 1, wherein mutation at the first genomiclocus is associated with cancer.
 13. The method of claim 12, wherein thefirst genomic locus is the KRAS gene.
 14. The method of claim 12,wherein the first genomic locus is the BRAF gene.
 15. The method ofclaim 1, wherein said sample is obtained from a human.
 16. The method ofclaim 15, wherein the sample is a stool sample.
 17. A reaction mixturecomprising: a) amplification reagents comprising a thermostablepolymerase, nucleotides, and: a first primer pair comprising a firstprimer and a second primer for amplifying a first target genomic locusfrom a nucleic acid sample; wherein the first primer of the first primerpair comprises: i. a 3′ terminal nucleotide that base pairs with a pointmutation in the first genomic locus; and ii. a nucleotide sequence thatis fully complementary to a sequence in the locus with the exception ofa single base mismatch within 6 bases of the 3′ terminal nucleotide; anda second primer pair comprising a first primer and a second primer foramplifying a second target genomic locus from a nucleic acid sample;wherein the first primer of the second primer pair comprises: i. a 3′terminal nucleotide that base pairs with a point mutation in the secondgenomic locus; and ii. a nucleotide sequence that is fully complementaryto a sequence in the locus with the exception of a single base mismatchwithin 6 bases of the 3′ terminal nucleotide; b) flap assay reagentscomprising: a flap endonuclease, a first FRET cassette and a first flapoligonucleotide that comprises a nucleotide that base pairs with thepoint mutation in the first genomic locus; and a second FRET cassetteand a second flap oligonucleotide that comprises a nucleotide that basepairs with the point mutation in the second genomic locus; c) thenucleic acid sample, wherein the nucleic acid sample comprises both wildtype copies of the first and second genomic loci and mutant copies ofthe first and second genomic loci, wherein the mutant copies of thefirst genomic locus have a point mutation relative to the wild typecopies of the first genomic locus and the mutant copies of the secondgenomic locus have a point mutation relative to the wild type copies ofthe second genomic locus; wherein the reaction mixture is characterizedin that it can amplify and detect the presence of the mutant copies ofthe genomic loci in the sample.
 18. The reaction mixture of claim 17,wherein the reaction mixture further comprises first invasiveoligonucleotide distinct from the first primer of the first primer pairand comprising a 3′ terminal nucleotide that base pairs with the pointmutation at the first genomic locus and/or a second invasiveoligonucleotide distinct from the first primer of the second primer pairand comprising a 3′ terminal nucleotide that base pairs with the pointmutation at the second genomic locus.
 19. A kit comprising: a) PCRreagents comprising: a first primer pair comprising a first primer and asecond primer, wherein the first primer comprises a 3′ terminalnucleotide that base pairs with a point mutation in a first genomiclocus and also comprises a nucleotide sequence that is fullycomplementary to a sequence in the first genomic locus with theexception of a single base mismatch within 6 bases of the 3′ terminalnucleotide; and a second primer pair comprising a first primer and asecond primer, wherein the first primer comprises a 3′ terminalnucleotide that base pairs with a point mutation in a second genomiclocus and also comprises a nucleotide sequence that is fullycomplementary to a sequence in the second genomic locus with theexception of a single base mismatch within 6 bases of the 3′ terminalnucleotide; b) flap assay reagents comprising: a first flapoligonucleotide that comprises a nucleotide that base pairs with thepoint mutation in the first genomic locus; and a second flapoligonucleotide that comprises a nucleotide that base pairs with thepoint mutation in the second genomic locus.
 20. The kit of claim 19,further comprising PCR and flap reagents for amplification and detectionof a control nucleic acid.